Femtosecond Three-Dimensional Imaging of Single-Protein with Hard X-ray Laser
The extremely intense pulses of X-ray free-electron lasers (XFELs) have enabled imaging of radiation-sensitive samples, such as macromolecular microcrystals, beyond radiation damage limits. These sources have the potential to deliver biomolecular single-particle imaging, similar to cryo-electron microscopy but without the need for cryo-fixation and with temporal resolution from femtoseconds to milliseconds. While this possibility was recognized before XFELs were built, the biological single-particle imaging work-flow has previously only been demonstrated on large virus particles. Based on decades of improvements in X-ray beam focusing, particle delivery, diffraction detection, and advanced analysis, here we demonstrate imaging of a single molecular complex, the giant-hemoglobin erythrocruorin (Ery) with X-ray laser pulses. Two-dimensional classes of diffraction patterns could be reconstructed to 15 Angstrom resolution, and 3D images to approximately 20 Angstrom, while the 3D merged intensity in reciprocal space extended beyond 20 Angstrom. The resolution discrepancy is likely due to heterogeneity caused by gas-phase compaction of the complexes. With increased throughput, this approach could be used to reveal in-situ structural details during mass spectrometry studies of biomolecules, while improvements in sample delivery may provide ultrafast snapshot imaging of biological single-particles in their native-state beyond the limitations of radiation damage.